High performance liquid chromatography (HPLC) is a technique that has been used for many years as a means of separating, identifying, purifying and quantifying components of often-complex mixtures. HPLC is an important tool used by biotechnological, biomedical, and biochemical research as well as in the pharmaceutical, cosmetics, energy, food, and environmental industries.
Conventional HPLC typically is performed using chromatographic columns with inside diameters (I.D.'s) in the range of 2.0-4.6 mm, 4.6 mm columns being a common standard. However, microcolumn LC, which is the most widely accepted term to describe liquid chromatography using packed columns having inside diameters of 2.0 mm or less, is gaining in popularity. Advantages of microcolumn LC include the ability to analyze smaller sample volumes, reduction of solvent usage, and enhanced mass sensitivity.
A relevant scaling factor in the design of LC systems is the square of the ratio of the inner diameters of the columns. The flow rate, the injection volume and the detection volume, among other parameters, all need to be decreased in proportion to this factor when the column I.D. is reduced in order to maintain chromatographic resolution.
Optical detectors are often used in HPLC systems to detect separated analytes within a fluid mixture following elution through a chromatographic column. Consistent with the scaling factor mentioned above, the volumes of optical detectors developed for use in conventional HPLC systems are too large to yield useful data when used in microcolumn LC systems. Such detectors would fail to adequately resolve components with small separations.
Another quantity that must be reduced to preserve performance when the column I.D. is reduced is the overall dispersion caused by the chromatographic system. Dispersion results in band broadening and the concomitant loss of chromatographic resolution. Variance is the second moment of the distribution function of a chromatographic peak and is the statistical quantity used to quantify dispersion. In the case of normal distributions, variance is the square of the standard deviation. The total variance of a peak is the sum of the variances resulting from both columnar and extra-columnar, or instrumental (injector, transfer lines, detector, etc.) sources. To preserve the separation resolution achieved by the column as the column I.D. is reduced, the extra-column variance must decrease by the ratio of the column inner diameters raised to the fourth power. While the variance contribution from instrumental sources is constant during a chromatographic run under the same experimental conditions, the variance due to the column is proportional to the square of the elution time. For this reason, the contribution of instrumental variance to total variance will be largest at early elution times. An acceptable amount of variance due to instrumental dispersion is considered to be about 10% of the column variance at the elution time of a non-retained peak (k=(t−tnr)/tnr=0, where k is the retention factor, t is the retention time of a given peak, and tnr is the retention time of a non-retained peak, which is the time it takes the mobile phase to flow from the injector, through the column, to the detector).
An example of the dramatic reduction in instrumental variance required to preserve the column efficiency in microscale HPLC systems can be derived by adapting information given in J. P. C. Vissers, “Recent Developments in Microcolumn Liquid Chromatography” J. Chromatogr. A 856 (1999) 117-143. Extrapolating from Vissers, the maximum acceptable variance due to instrumental dispersion (10% of the column variance) at k=0 for a column having a 1.0 mm inner diameter and a 15.0 cm length is 90,600 n12, whereas for a column having a 300 μm inner diameter, the maximum acceptable variance due to instrumental dispersion is about 740 n22 for equivalent experimental conditions.
Techniques known in the art, such as the “stacking” of analytes at the head of a column prior to gradient elution, can be used to minimize the contribution of instrumental variance from components prior to the column. However, contribution from instrumental sources following the column, notably the detection cell, cannot be reduced in this manner.
Accordingly, there is a need in the art for a microfluidic detection device having reduced dispersion that can perform photometric measurements on a flowing liquid sample and which is suitable for use in microcolumn LC systems.